基于胆汁酸代谢网络分析异甘草素通过氧化/化学应激防御通路Nrf2/ARE的保肝作用

Evaluation on Hepatoprotection in Nrf2/ARE Oxidation/Chemical Stress Defense Pathway Caused by Isoliquiritigenin Based on Analysis of Bile Acids

目的本实验通过HPLC-MS/MS测定小鼠肝脏中16种胆汁酸浓度,从而运用胆汁酸代谢网络分析异甘草素(isoliquiritigenin,Iso)在氧化/化学应激防御通路Nrf2/ARE中的保肝作用。方法课题组前期通过荧光素酶报告基因法筛选甘草提取物中Nrf2/ARE信号靶向分子的实验,发现异甘草素显著激活了Nrf2驱动的报告基因表达,可以用于氧化/化学应激防御通路Nrf2/ARE的肝脏保护研究。使用SIMPCA-P软件对肝脏中胆汁酸含量测定结果进行主成分分析、偏最小二乘法-判别分析。结果野生型小鼠组(Nrf2+/+)、Nrf2基因敲除小鼠组(Nrf2-/-)、异甘草素干预的野生型小鼠组(Iso Nrf2+/+)均与异甘草素干预的Nrf2基因敲除小鼠组(Iso Nrf2-/-)区分明显,且(Iso Nrf2-/-)组间较分散,提示Iso通过Nrf2/ARE通路产生抗氧化应激产物,促使体内胆汁酸外排,降低肝细胞和血液中胆汁酸水平,从而保护肝脏。再运用SPSS19.0统计学软件以Hotelling T2检验方法分析数据,结果显示,4组相互比较间有统计学意义的胆汁酸为:熊去氧胆酸(UDCA)、鹅去氧胆酸(CDCA)、胆酸(CA)、牛磺脱氧胆酸(TDCA)、去氧胆酸(DCA)、牛磺胆酸(TCA),表明这6个胆汁酸可作为评价Iso诱导Nrf2后产生保护肝脏作用的生物标识物。结论本实验为进一步深入探讨甘草保肝作用及解毒机制提供了参考。

OBJECTIVE To evaluate hepatoprotection in Nrf2/ARE oxidation/chemical stress defense pathwaycaused by isoliquiritigenin （Iso） based on analysis of bile acids using profile of bile acids. METHODS High performance liquid chromatogra- phy coupled with quadrupole mass spectrometry （HPLC-MS/MS） was applied to determine the contents of all kinds of endogenous bile acids including free bile acids, taurine conjugates and glycine conjugates. By the luciferase reporter gene assay for screening Nrf2-ARE signal targeting molecular experiments in liquorice extract,isoliquiritigenin was capable of markedly activating Nrf2-driven gene expres- sion, therefore it was used to evaluate hepatoprotection in Nrf2/ARE oxidation / chemical stress defense pathway. RESULTS Based on the analysis using principle components analysis （PCA） ,partial least square-discriminant analysis （PLS-DA）, Nrf2＋/＋, Nrt2-/- and Iso Nrt2 ＋/＋ groups could be distinguished from their Iso Nrf2 -/- group, which suggested that the variance of the contents of bile acids could evaluate hepatoprotection caused by Iso. Bile acids of ursodeoxycholic acid （ UDCA）, chenodeoxycholic acid （ CDCA）, cholic acid （ CA）, sodium taurodeoxycholate hydrate （ TDCA）, deoxycholic acid （ DCA）, taurocholic acid （TCA） proved to be impor- tant corresponds to Iso induced liver protection according to analysis of partial least square-discriminant analysis （PLS-DA） and the statistical analysis showed that there were significant differences among the four groups. It indicated that UDCA, CDCA, CA, TDCA, DCA and TCA could be considered as sensitive biomarkers of Iso induced liver protection. CONCLUSION This work can provide the base for the further research on the evaluation and mechanism of hepatoprotection caused by liquorice.